Advances in the field of molecular biology over the last two decades have enabled the detection of specific nucleic acid sequences in test samples taken from a patient or other subject. Such test samples include serum, urine, feces, tissue, saliva, cerebrospinal fluid, amniotic fluid, and other bodily fluids. Detection of specific nucleic acid sequences can be used to identify genetic disorders or diseases, as well as the presence of pathogenic bacterial and viral associated diseases in humans. The presence of specific genes can also be used to obtain other pertinent genetic information, such as the presence of genes coding for antigens responsible for graft rejection, as well as genetic information used in cancer and oncogene testing and in forensic medicine.
The most common techniques for detecting a particular gene sequence utilize a phenomenon known as nucleic acid hybridization. Hybridization refers to a particular type of recombination, or annealing, of a single-stranded nucleotide sequence with a complementary sequence to form a double-stranded nucleic acid, or duplex, through non-covalent bonding. Where the original strands of a normally double-stranded DNA (deoxyribonucleic acid) molecule recombine, the recombination process is referred to as renaturation. Where molecular mixing occurs, the process is referred to as hybridization. Hybridization can also occur with RNA (ribonucleic acid) which is frequently found in nature in both a double-stranded and a single-stranded form. DNA:RNA hybrids can be formed through hybridization, as well as DNA:DNA and RNA:RNA hybrids.
In typical hybridization techniques, a single-stranded probe sequence is used to seek out the gene or nucleic acid sequence of interest by annealing, or hybridizing, to the single-stranded form of the target nucleic acid sequence. Where the nucleic acid in a test sample is double-stranded, as is typically the case with naturally occurring DNA, the DNA must be denatured, or rendered single-stranded, before any form of recombination can take place. The target sequence is a selected portion of the nucleic acid sequence being sought which is uniquely characteristic of that target sequence. The probe is selected to be a single-stranded nucleotide sequence complementary to the target nucleotide sequence of the gene being sought. This oligonucleotide probe is marked with a detectable label and brought into contact with the denatured nucleic acid (DNA or RNA) from a test sample.
Until recently, radioactive isotopes of atoms such as hydrogen (3H), phosphorus (32P), or iodine (125I) were primarily used for probe labels. Radioactive compounds such as these, however, suffer from many drawbacks, including the necessity of incorporating extensive safety precautions into assay protocols, the need for expensive equipment and special waste treatment procedures, as well as high usage costs due to the instability of the radioactive materials. As a result, there have been increasing efforts in recent years to develop alternative labeling schemes not having the drawbacks of radioactive isotope labels. Consequently, non-isotopically labeled probes are currently used in addition to radioactive labels, although non-radioactive labels are preferred in a clinical setting.